Mechanistic Studies of Cu(II) Binding to Amyloid- Peptides and the Fluorescence and Redox Behaviors of the Resulting Complexes Nakul C. Maiti, Dianlu Jiang, Andrew J. Wain, Sveti Patel, Kim L. Dinh, and Feimeng Zhou* Department of Chemistry and Biochemistry, California State UniVersity, Los Angeles, California 90032 ReceiVed: March 7, 2008; ReVised Manuscript ReceiVed: April 28, 2008 Due in large part to the lack of crystal structures of the amyloid- (A) peptide and its complexes with Cu(II), Fe(II), and Zn(II), characterization of the metal-A complex has been difficult. In this work, we investigated the complexation of Cu(II) by A through tandem use of fluorescence and electron paramagnetic resonance (EPR) spectroscopies. EPR experiments indicate that Cu(II) bound to A can be reduced to Cu(I) using sodium borohydride and that both A-Cu(II) and A-Cu(I) are chemically stable. Upon reduction of Cu(II) to Cu(I), the A fluorescence, commonly reported to be quenched upon A-Cu(II) complex formation, can be regenerated. The absence of the characteristic tyrosinate peak in the absorption spectra of A-Cu(II) complexes provides evidence that the sole tyrosine residue in A is not one of the four equatorial ligands bound to Cu(II), but remains close to the metal center, and its fluorescence is sensitive to the copper oxidation state and perturbations in the coordination sphere. Further analysis of the quenching and Cu(II) binding behaviors at different Cu(II) concentrations and in the presence of the competing ligand glycine offers evidence supporting the operation of two binding regimes which demonstrate different levels of fluorescence recovery upon addition of the reducing agent. We provide results that suggest the fluorescence quenching is likely caused by charge transfer processes. Thus, by using tyrosine to probe the coordination site, fluorescence spectroscopy provides valuable mechanistic insights into the oxidation state of copper ions bound to A, the binding heterogeneity, and the influence of solution conditions on complex formation. Introduction Alzheimer’s disease (AD) is a progressive neurological disorder, most commonly associated with dementia in the elderly. Although not fully understood, substantial evidence indicates that the disease is caused by neurotoxic assemblies of amyloid -peptides (A). 1,2 The general hypothesis is that A peptide first coalesces to form small, soluble oligomers, 3–5 followed by reorganization and assembly into long, insoluble, and often twisted, thread-like fibrils. In addition to numerous biochemical events, environmental factors such as the presence of metal ions are also purported to be involved in the A aggregation process and neuropathogenesis of AD. 6 It has been shown that metal ions such as Cu(II), Fe(II), and Zn(II) bind to A peptide, 6–12 and the resulting complexes have been linked with the generation of reactive oxygen species (ROS). 10,13 Recent investigations have indicated that the segment HDS- GYEVHH of A (Figure 1), located in the hydrophilic domain near the N-terminus of the peptide, plays a crucial role in metal coordination. 6–11 Electron paramagnetic resonance (EPR) mea- surements suggest that, at neutral pH, three imidazole nitrogens (from His-6, His-13, and His-14) and one oxygen-containing group participate in coordination to Cu(II) as the four ligands in a square-planar geometry. 9 Because of the lack of crystal structures of A peptides and their strong tendency to aggregate, it has been difficult to probe the coordination chemistry. As a result, the identity of the fourth ligand has been elusive, though possible candidates include the N-terminal amine, 9,14 carboxylate groups from aspartic or glutamic acid residues (Asp-1 or Glu-3), 8,14 and the sole tyrosine residue, Tyr-10. 10,15 The involvement of the tyrosine residue in particular appears to be in debate. 8–11,16 For instance, direct evidence ruling out Tyr-10 as a possible ligand was presented on the basis of EPR experiments in which an oxygen-bearing ligand was still found to be present in A mutants lacking the Tyr-10 residue. 8 On the other hand, broadening of the tyrosine proton resonance in the 1 H NMR spectrum of A-Cu(II) complexes has been interpreted by Ma et al. as indirect evidence that Tyr-10 is bound to the copper center. 16 However, Faller’s group measured no such broadening in the 1 H NMR spectra 17 and Kowalik- Jankowska and co-workers reported that the phenolic pK a of Tyr-10 is unchanged by the presence of copper, suggesting that its participation in binding is unlikely. 14 These apparent inconsistencies have remained unresolved. Tyr-10 is located in close proximity to the three histidine residues (Figure 1) that are believed to be involved in metal coordination, 6 rendering it sensitive to metal-induced chemical changes. Tyr-10 yields the intrinsic fluorescence of A and has been frequently used to investigate the coordination of transition metal ions to A. 8,9,16,18,19 It is commonly known that Cu(II) binding to A results in varying degrees of fluorescence quenching. However, the mechanism of fluorescence quenching has not been investigated. We show in this work that the oxidation state and coordination geometry of the bound copper ion both affect the A fluorescence behavior, indicating a close association between the Tyr-10 residue and the copper ion. Absorption spectroscopy reveals that Tyr-10 does not become a tightly bound ligand to Cu(II), but is influenced by charge transfer processes which may rationalize the fluorescence quenching observations. By changing the oxidation state of the copper center and studying the resultant fluorescence spectra, we obtained further evidence that may support the existence of two species of or two different binding regimes in A-Cu(II) * Corresponding author. Phone: (323) 343-2390. Fax: (323) 343-6490. E-mail: fzhou@calstatela.edu. J. Phys. Chem. B 2008, 112, 8406–8411 8406 10.1021/jp802038p CCC: $40.75  2008 American Chemical Society Published on Web 06/21/2008